This invention relates to novel room-temperature process for obtaining calcium phosphate, in particular hydroxyapatite, microspheres and coatings with encapsulated drugs, proteins, genes, DNA for therapeutical use. The coatings and microspheres are designed to perform a defined biological function related to drug delivery, such as gene therapy through gene delivery. A novel method for encapsulation, and subsequent controlled release of therapeutically active agents from such biofunctional coatings and microspheres is disclosed. Such coatings and microspheres are useful for side effects-free, long-term, targeted, controlled release and delivery of drugs, proteins, DNA, and other therapeutic agents.
Rapid progress in the human genome project promises that diseases that could not be treated before can be curable in near future. The expectation is that the trial-and-error era of fighting illnesses by addressing the symptoms is coming to an end. Consequently, the issue of drug and gene release control becomes increasingly critical. Currently, many types of new drugs, or genes, have to be administered by daily injections, or even several times per day. An entirely new approach to drug delivery is therefore necessary to fully utilize the advantages of new drugs resulting from the genome project. Slow but steady release of drugs is sought in treatment of many diseases, from cancer and Parkinson""s disease, to hormonal treatment of obesity where directly administered hormones reside in human body only for short period of time. In the past, polymeric materials have been used for drug delivery control and enjoyed substantial clinical success for certain drug systems. The need for alternative inorganic drug delivery systems, offering more flexibility in drug-carrier system selection, bioresorption and release control, hydrophobic/hydrophilic property control, and negligible side effects, is just emerging. Hydroxyapatite (HA) matrix for drug encapsulation, being already the principal inorganic component of bone, offers entirely new perspectives for drug delivery systems.
Hydroxyapatite Ceramics, Ca10(PO4)6(OH)2, belong to a large class of calcium phosphate (CaP) based bioactive materials used for a variety of biomedical applications, including matrices for drug release control [M. Itokazu et al Biomaterials, 19,817-819,1998; F. Minguez et al Drugs Exp. Clin. Res., 16[5], 231-235, 1990; W. Paul and C. P. Sharma, J. Mater. Sci. Mater. Med., 10, 383-388,1999]. Other members of the CaP family, such as dicalcium phosphate CaHPO42H2O or tricalcium phosphate Ca3(PO4)2, have also been used for similar purposes. The CaP family of materials has been long recognized as having highest degree of biocompatibility with human tissue.
Calcium Phosphate Cements (CPC) were reported in a binary system containing tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrate (DCPA) [L. C. Chow et al. J. Dent Res., 63, 200,1984]. The CPC advantages of self-setting and apatitic phase, e.g. HA, as an end product led to applications such as bone replacements and reconstruction, and also drug delivery [M. Dairra, et al. Biomaterials, 19 1523-1527, 1998; M. Otsuka, et al. J. of Controlled Release 43(1997)115-122, 1997; Y. Tabata, PSTT, Vol.3, No.3, 80-89, 2000; M. Otsuka, et al. J. of Pharm. Sci. Vol.83, No.5, 1994]. CPC is typically formulated as a mixture of solid and liquid components in pertinent proportions, which react to form the apatite. The physicochemical reactions that occur upon mixing of the solid and liquid components are complex, but dissolution and precipitation are the primarily mechanisms responsible for the final apatite formation [C. Hamanish et al J. Biomed. Mat. Res., Vol.32, 383-389,1996; E. Ferandez et al J. Mater. Sci.Med.10,223-230, 1999]. The reaction pathway in most CPC systems does not lead to stoichiometric HA, but rather calcium-deficient Ca10xe2x88x92x(HPO4)x(PO4)6xe2x88x92x(OH)2xe2x88x92x, similar to that found in bone. The process parameters, such as Ca/P ratio, powder/liquid ratio, seeds concentration and type, nature of reagents, control the final properties, such as phase content, porosity, strength, setting time, phase transformation kinetics, and microstructure of CPC-derived hydroxyapatite (CPC-HA). Bermudez et al [J. Mat. Sci. Med. 4, 503-508,1993; ibid 5, 67-71, 1994] correlated the compressive strength of CPC vs starting Ca/P ratio in the systems of monocalcium phosphate monohydrate (MCPM) and calcium oxide. The optimum Ca/P ratio was found in a range of 1.25-1.45. Brown et al. [J. Am. Cerm. Soc. 74[5] 934-40,1991] found that the kinetics of HA formation at low temperatures in DCP/TTCP system are initially controlled by the surface area of the reactants, and eventually by diffusion. These process variables will be used in the present project to control crystallinity, and thus resorption and drug release rate from the HA microspheres.
Biomimetic Deposition of HA films at room temperature (BM-HA) was used for a variety of biomedical applications, including drug delivery [H. B. Wen et al, J. Biomed. Mater. Res., 41, 227-36,1998; S. Lin and A. A. Campbell, U.S. Pat. No. 5,958,430, 1999; D. M. Liu et al J. Mater. Sci. Mater. Med., 5, 147-153,1994; K. de Groot et al, J. Biomed. Mater. Res., 21, 1375-1381,1987]. This forming mechanism is driven by supersaturation of Ca2+ and PO43xe2x88x92, under appropriate solution pH, where HA is the most stable phase. The apatitic crystals form through nucleation and growth, and may incorporate biologically active species, such as antibiotics, anti-cancer drugs, anti-inflammatory agents, etc. The deposition rates are however small for BM-HA, generally in the range of 0.05-0.5 xcexcm/h, and high concentration dosage of drug is difficult to achieve. Therefore, stand-alone BM-HA is not suitable if the goal is to form films in excess of 1 xcexcm, but may be appropriate as an additional encapsulating film, on top of the porous HA structure saturated with the drug material. This is especially critical for orthopedics where high concentration of antibiotics is required at bone-HA interface to prevent acute inflammation in early after-operation stages. Furthermore, the physiological solutions for BM-HA formation are naturally water-based, which makes impossible to encapsulate hydrophobic bioactive agents into BM-HA coatings.
Sol-Gel Deposition of HA (SG-HA) films at elevated temperatures (375-500xc2x0 C.) was disclosed previously by D. Liu and T. Troczynski in U.S. patent application Ser. No. 09/563,231, filed May 2, 2000, the subject matter of which is incorporated herein by reference. Sol-gel (SG) processing of HA allows molecular-level mixing of the calcium and phosphor precursors, which improves chemical homogeneity of the resulting calcium phosphate. The versatility of the SG method opens an opportunity to form thin film coatings in a simple, mild, relatively low-temperature process. The crystallinity of the calcium phosphate phase obtained through the novel inventive process by D. Liu and T. Troczynski can be enhanced by appropriate use of water treatment during processing. Variation of Ca/P ratio in the sol-gel precursor mix allows one to obtain other than calcium phosphate phases, for example, hydroxyapatite, dicalcium phosphate, tricalcium phosphate or tetracalcium phosphate. The use of SG-HA thin ( less than 1 xcexcm) dense highly crystalline films to nucleate and grow thick ( greater than 10 xcexcm) CPC-HA porous, low-crystallinity (amorphous) films for drug encapsulation and release is hereby disclosed.
Problems With Drug Delivery in Vivo are related to toxicity of the carrier agent, the generally low loading capacity for drugs as well as the aim to control drug delivery resulting in self-regulated, timed release. With the exception of colloidal carrier systems, which support relatively high loading capacity for drugs, most systems deliver inadequate levels of bioactive drugs. In terms of gene delivery, to date the most efficient, though least safe methods of delivery are through viral mediated gene transfer. It is highly inefficient method, and is faced with even greater problems than the delivery of drugs due to the hydrophilic and labile nature DNA oligos. The problems with delivery of genes or antisense oligos originate from the rapid clearance of plasmid DNA or oligos by hepatic and renal uptake as well as the degradation of DNA by serum nucleases [Takura Y, et al. Eur. J. Pharm. Sci. 13 (2001) 71-76]. These effects have been observed for both in situ and intravenous delivery. For example it was estimated that more than half of the intravenous or in situ delivered naked plasmid DNA was cleared from the tumor site within the first two hours following intratumoral injection [Ohkouchi, K., et al Cancer Res. 50, (1996) 1640-1644 and Imoto, H., et al. Cancer Res. 52, (1992), 4396-4401]. Even after clearance, only a small percentage of the remaining DNA or oligos make their way to the cytoplasm or nucleus of the target cell. The membrane permeability of naked DNA and especially oligos is virtually nonexistent, due to their polyanionic nature. For this reason, their uptake through the endosomal compartment is associated with a severe drop in pH and degradation. Finally, many of the genes delivered have to be transported and sometimes incorporated in the genome of the target cell for stable expression. This makes very difficult gene transfer in vivo. In addition, successful controlled release is still problematic as for most applications (with the exception of naked DNA vaccines) it is desirable to have a prolonged expression of the gene of interest to ameliorate a particular medical condition. In most applications anywhere from a few weeks to several months are desired for the expression of a certain gene product.
Drug Encapsulation in HA has been achieved in the past by simple post-impregnation of a sintered, porous HA ceramic [K. Yamamura et al, J. Biomed. Mater. Res., 26, 1053-64,1992]. In this process, the drug molecules simply adsorb onto surface of the porous ceramic. The drug release is accomplished through desorption and leaching and the drug to the surrounding tissue after exposure to physiological fluid. Unfortunately, most of the adsorbed drug molecules release from such system in a relatively short period of time. Impregnation of drug material into porous sintered calcium phosphate microspheres has been reported in patent literature. xe2x80x9cSlow releasexe2x80x9d porous granules are claimed in U.S. Pat. No. 5,055,307 [S. Tsuru et al, 1991], wherein the granule is sintered at 200-1400 C and the drug component impregnated into its porosity. xe2x80x9cCalcium phosphate microcarriers and microspheresxe2x80x9d are claimed in WO 98/43558 by B. Starling et al [1998], wherein hollow microspheres are sintered and impregnated with drugs for slow release. D. Lee et al claim poorly crystalline apatite [WO98/16209] wherein macro-shapes harden and may simultaneously encapsulate drug material for slow release. It has been suggested to use porous, composite HA as a carrier for gentamicin sulfate (GS), an aminoglycoside antibiotic to treat bacterial infections at infected osseous sites [J. M. Rogers-Foy et al, J. Inv. Surgery 12 (1997) 263-275]. The presence of proteins in HA coatings did not affect the dissolution properties of either calcium or phosphorus ions and that it was solely dependent on the media [Bender S. A. et al. Biomaterials 21 (2000) 299-305].
The group of Kobe University lead by Prof. M. Otsuka performed series of investigations of drug encapsulation in self-setting calcium phosphate cements derived from tetracalcium phosphate and dicalcium phosphate [J. Contr. Rel. 43 115-122 1997; ibid 52 281-289 1998; J. Pharm. Sci. 83 611-615, 259-263, 255-258, 1994]. The cement was shaped with in-situ drug encapsulation, into 15 mm diameter macro-pellets and drug (indomethacin) release monitored over up to 3 weeks period. It was concluded that the cement-drug delivery system, shaped in-situ into surrounding bone tissue, may be an excellent way to treat localized bone infections with high therapeutic effectiveness. The advantage of HA for drug delivery is that side effects have never been a concern for hydroxyapatite materials [Y. Shinto et al, J. Bone Jt. Surg., 74B4, 600-4,1992]. Calcium phosphatexe2x80x94biodegradable polymer blends were also investigated as possible vehicles for drug delivery [I. Soriano and C. Evora, J. Contr. Rel. 68 121-134 2000]. Prolonged drug release (up to 10 weeks) was obtained for the composites coated with hydrophobic polymer coatings. A group from University of Pennsylvania [Q. Qiu et al J. Biomed Mat Res. 52 66-76 2000] recently processed polymer-bioactive glass-ceramic composite microspheres for drug delivery. Porous calcium phosphate ceramics were impregnated with bone marrow cells [E. Kon et al, J. Biomed Mat. Res. 49 328-337 2000] and with human bone morphogenetic protein [I. Alam et al J. Biomed Mat. Res. 52 2000].
S. Takenori et al., in U.S. Pat. No. 5,993,535 (and accompanying EP0899247), disclosed a calcium phosphate cement comprising tetracalcium phosphate and calcium hydrogen phosphate polysaccharide as main components. It needed 24 hours incubation at 37xc2x0 C. for conversion of hydroxyapatite. T. Sumiaki et al., in U.S. Pat. No. 5,055,307, disclosed slow release drug delivery granules comprising porous granules of a calcium phosphate compound having a ratio of Ca to P of 1.3 to 1.8, a porosity of 0.1 to 70%, a specific surface area of 0.1 to 50 m2/g and a pore size of 1 nm to 10 xcexcm. The granules were fired at a temperature of 200 to 1400xc2x0 C., and a drug component impregnated in pores of the granules, and a process for producing the same. S. Gogolewski, in WO00/23123, disclosed the hardenable ceramic hydraulic cement comprising a drug to be delivered to the human or animal body upon degradation or dissolution of the hardened cement. However, conversion of CPC to achieve HA needed 40 hours. L. Chow et al., in U.S. Pat. No. 5,525,148, disclosed calcium phosphate cements, which self-harden substantially to hydroxyapatite at ambient temperature when in contact with an aqueous medium. More specifically the cements comprise a combination of calcium phosphates other than tetracalcium phosphate with an aqueous solution adjusted with a base to maintain a pH of about 12.5 or above and having sufficient dissolved phosphate salt to yield a solution mixture with phosphate concentration equal to or greater than about 0.2 mol/L. However, major disadvantages of the processing are that high pH ( greater than 12.5), which could denature most of drugs, proteins and DNA, so the process is not suitable for drug encapsulation vehicles. C. Rey et al., in WO9816209, disclosed a synthetic, poorly crystalline apatite calcium phosphate containing a biologically active agent and/or cells, preferably tissue-forming or tissue-degrading cells, useful for a variety of in vivo and in vitro applications, including drug delivery, tissue growth and osseous augmentation. However, the ratio of Ca/P was limited less than 1.5, and the authors did not disclose how to fabricate the microspheres and coatings.
The physical characteristics, i.e. shape, of the hydroxyapatite ceramic, also have significant impact on the tissue response. Among different possible shapes, hydroxyapatite granules facilitate not only surgical operations but also benefit the tissue growth after implantation by creating relatively large inter-granular pores allowing invasion by the host tissue. On this basis, the use of drug-containing HA granules has enhanced therapeutic effect in practical clinical/biomedical applications.
While the above studies describe the dissolution of porous HA to release a soluble extracellular acting bioactive ingredient through desorption and leaching, this disclosure aims also intracellular delivery of genes, drugs or proteins using resorbing HA microcarriers. The key observation is that HA particles were found inside macrophages at the interface of HA-coated hip implants [Bauer T. W. et al. J. Bone Joint Surg. Am. 73-A (1991) 1439-1452]. Consequently it was proposed that phagocytes take up and solubilize HA particles [Evans R. W. et al. Calcif. Tissue. Int. 36 (1984) 645-650]. This may be therefore one of the principal mechanisms of intracellular delivery of genes through use of HA coatings and microspheres, without the need for use viral mediated gene transfer.
We hereby disclose a process through which hydroxyapatite can be engineered to function as an efficient vehicle for drug delivery from coatings or self-supported microspheres. The process relates to calcium phosphate (CaP), in particular hydroxyapatite (HA) microspheres and coatings capable to encapsulate any type of drug or protein which can be dispersed in organic liquids or water. The process starts with calcium phosphate cement (CPC) slurry, followed by incubation to precipitate HA phase within the microsphere or within the coating. By adding drugs and proteins into colloidal suspension (CPC slurry) of the microsphere or coating precursors, a direct, in-situ encapsulation, and subsequent controlled release of the therapeutically active agents from the apatite microspheres is achieved. The varying degree of crystallinity of the microspheres is used to control and customize their resorption process in body fluids, and thus the rate of drug release.
Despite the fact that several techniques were investigated in the past in use of static macro-components of CaP for drug delivery, none of them allows processing of fast setting functionally gradient cement microspheres and coatings with in-situ drug encapsulation. Consequently we disclose hereby a new, safe and inexpensive way to deliver drugs, proteins, genes and antisense oligos in vivo. In this process calcium phosphate coatings and microspheres are obtained through dissolution-precipitation mechanism similar to setting of calcium phosphate cements (CPC). This process is used for deposition of adhesive, thick (typically of 10-1000 xcexcm thickness) HA films and for making HA microspheres (typically of 10-1000 xcexcm diameter).
Calcium phosphate cements, like most other cements (e.g. Portland cement) release heat of hydration when hydrating and setting. When setting fast (which is an important requirement in certain applications), this heat may not dissipate fast enough and temperature of cemented area increases, sometimes to a level high enough to damage, e.g. crack, the cement. More importantly, if the cement is applied in human body, e.g. as part of an implant, this temperature increase may damage the surrounding biological constituents such as cells, proteins, and enzymes when the heat is rapidly dissipated upon setting. The novel cement system disclosed here, experiences a relatively mild temperature rise during setting, i.e. from room temperature, xcx9c20xc2x0 C., to a maximum near body temperature, xcx9c37xc2x0 C. This cement also exhibits excellent mechanical properties. For example, its compressive strength is generally greater than 10 MPa, and in certain compositions,  greater than 30 MPa.
This CPC slurry is a mixture of acidic calcium phosphates, such as monocalcium phosphate (MCP) anhydrate or dihydrate, and basic calcium hydroxide, together with a small amount of other inorganic ingredients, such as apatite seeds. This mixture is thoroughly mixed, e.g. using ball milling. The mixture, which is suspended in an inert liquid medium (e.g. alcohols) or a liquid mixture of the inert medium and a small amount of phosphating liquid, has a starting Ca/P ratio in the range of 1.2-1.67 and a solid concentration of 30-70 vol %, before the shaping process is being carried out (i.e. filling the cavity with the cement or shaping a drug-impregnated microsphere). The shaping procedures can be optimized upon control of the setting time within 2-40 minutes.
The resulting cement exhibits an apatitic phase (HA) with either an acicular grain or plate-like grain morphology, depending upon processing parameters. A nano-structured cement can also be synthesized and is used particularly for encapsulating drugs, antibiotics, proteins, and enzymes. The apatite phase develops within 1-6 h of incubation within the CPC system disclosed. Fast formation of the apatite phase is advantageous for an early biological response with the surrounding host tissue. A fast setting (2-40 min) and hardening time (less than 6 hours) benefits enormously clinical applications of the cement. Controllable crystallinity, from amorphous (easily resorbable) to highly crystalline (i.e. stable in physiological environment), of the final apatitic structure allows application-oriented customization of the cement. Fine (nano-to-submicron) starting ingredients allow final microstructure to be well controlled, allowing high strength of the final body. Nano-structure of the novel CPC offers great advantage for encapsulating drugs, as well as for their controlled release. Apatite microspheres can be easily fabricated using the novel CPC formulation, with size ranging from 10-1000 microns, using simple spray-drying equipment. Control of the setting and hardening process of the cement microspheres allows a variety of biomedical or clinical applications, e.g., injection-packing, and in-situ hardening for repair, restoration, augmentation of defective tissues, and many others.
The disclosed process can be utilized to synthesize HA ceramics of different physical forms, at room temperatures. The following applications are targeted: granules or bulk shapes for artificial bone filler/bone reconstruction; room-temperature processed coatings (on Ti and other alloys), with drug incorporation; organic/inorganic composites, including HA in combination with other materials such as biopolymers and ceramics, and proteins to form nano- and bio-composites with controlled drug release function and/or bone growth control functions.
We disclose here a novel approach to achieve a multi-layer, functionally gradient HA coatings or microspheres. That is, several layers of HA coatings are deposited using different techniques, with different functions assigned to any particular layer. In particular, we describe an in-situ drug encapsulation within the functionally gradient HA coatings processed by combining elevated-temperature sol-gel processing with room-temperature precipitation processing. As a result of this protocol, a 10-30 xcexcm thick, nano-to-submicron porous HA matrix, firmly attached to a metallic substrate through a 0.6-0.8 xcexcm thin intermediate HA film is achieved. The thin sol-gel HA film (SG-HA) develops good bond with the underlaying substrate at the 400 C. processing temperature, and acts as an intermediate nucleation/bonding layer between the underlying substrate and the HA thick film matrix. The CPC-HA thick film matrix is deposited at room-temperature on the thin SG-HA film by dip coating in non-aqueous suspension of monocalcium phosphate and calcium hydroxide precursor powders. The drug material is dispersed in the suspension and initially deposited within the pores of the precursor powder film. Subsequently, a porous thick film apatite is formed under physiological environment, through dissolution-precipitation process. The thin SG-HA film acts as a seeding template allowing nucleation and growth of the thick film apatite crystals, and therefore imparts sufficient interfacial strength to the HA matrix. This room-temperature coating process, associated with an in-situ encapsulation of drugs, can be applied onto variety of metallic or non-metallic substrates, providing potential advantages in various biomedical/biochemical applications.
The microspheres are self-supporting granules produced through spray-drying and incubation process of CPC-HA precursors slurry, using sythesis routes and chemistry much the same as for the coatings. Further details of the invention are provided below.
A room-temperature process for deposition of adhesive, thick hydroxyapatite films on metallic substrates through dissolution-precipitation mechanism similar to setting of calcium phosphate cements (CPC) is disclosed. Such nanoporous, semi-amorphous 10-1000 xcexcm thick film is rapidly grown on the underlying crystalline thin film of hydroxyapatite deposited through sol-gel process. A direct, in-situ encapsulation, and subsequent controlled release of therapeutically active agents from the apatite coatings has been achieved. Slight modification of similar process leads to formation of medicated HA microspheres of 10-1000 xcexcm diameter. Both the coatings and microspheres are designated for side-effects free, long-term, targeted, controlled release and delivery of drugs, proteins, DNA, and others.
The invention is directed to a process for preparing a calcium phosphate phase which comprises: (a) dispersing a homogenization of monocalcium phosphate Ca(H2PO4)2 and calcium hydroxide Ca(OH)2 precursors in a substantially water-free medium to form a paste or viscous slurry of precursors; (b) drying the precursors slurry; (c) admixing a water-based solution of phosphate ions in the slurry to produce a mixture; and (d) incubating the resulting mixture in a humid environment at a temperature of 20-50xc2x0 C. to dissolve the precursors and precipitate a calcium phosphate phase.
The calcium phosphate phase can be hydroxyapatite. The Ca/P atomic ratio in the precursors mixture can be in the range of 1.2-1.67 and the mixture can have a solids concentration of 30-70 vol %. The substantially water-free medium can be an alcohol. The alcohol can be ethyl alcohol, methyl alcohol or propyl alcohol. The water-based solution of phosphate ions can be sodium phosphate and the humid environment can be 50% to 100% Relative Humidity.
The precursors slurry can be dried in a prescribed shape. The shape can be a pellet, a coating, a microsphere, or a dental cavity or bone cavity fill. A crystalline hydroxyapatite powder can be added to the precursors slurry to promote crystallization of hydroxyapatite calcium phosphate phase during the incubation period. A therapeutically active material can be added to the precursors slurry for encapsulation during crystallization of the hydroxyapatite calcium phosphate phase during the incubation period. The therapeutically active material can be a drug, a protein, a gene or DNA.
The slurry of precursors can be deposited and dried on the surface of a calcium phosphate substrate. The calcium phosphate substrate can be a thin film of hydroxyapatite coating deposited on a metallic substrate using sol-gel process. The precipitated calcium phosphate phase can be exposed to a solution supersaturated with Ca2+ and PO43xe2x88x92ions to provide a coating of biomimetic hydroxyapatite film on the precipitated calcium phosphate phase.
The slurry of precursors can be atomized and spray-dried to obtain microspheres of the precursors. The microspheres of the precursors can be exposed to a water-based solution of phosphate ions and incubated in a humid environment at a temperature of 20-50xc2x0 C. to promote dissolution of the precursors and precipitation of calcium phosphate phase. The microsphere containing the precipitated calcium phosphate phase can be exposed to a solution supersaturated with Ca2+ and PO43xe2x88x92ions to provide a coating of biomimetic hydroxyapatite film on the precipitated calcium phosphate phase. The precipitated calcium phosphate phase can be exposed to a solution polymer to form a polymer film on the microsphere surface.